Gap junctions mediate electrical signaling and ensuing cytosolic Ca2+ increases between chromaffin cells in adrenal slices: A role in catecholamine release

Abstract

In adrenal chromaffin cells, a rise in cytosolic calcium concentration ([Ca(2+)]i) is a key event in the triggering of catecholamine exocytosis after splanchnic nerve activation. Action potential- or nicotine-induced [Ca(2+)]i transients are well described in individual chromaffin cells, but whether they remain spatially confined to the stimulated cell or propagate to adjacent cells is not yet known. To address this issue, the spatiotemporal organization of electrical and associated Ca(2+) events between chromaffin cells was investigated using the patch-clamp technique and real-time confocal imaging in rat acute adrenal slices. Spontaneous or electrically evoked action potential-driven [Ca(2+)]i transients were simultaneously detected in neighboring cells. This was likely attributable to gap junction-mediated electrotonic communication, as shown by (1) the bidirectional reflection of voltage changes monitored between cell pairs, (2) Lucifer yellow (LY) diffusion between cells exhibiting spontaneous synchronized [Ca(2+)]i transients, and (3) the reduction of LY diffusion using the uncoupling agent carbenoxolone. Furthermore, transcripts encoding two connexins (Cx36 and Cx43) were found in single chromaffin cells. This gap junctional coupling was activated after a synaptic-like application of nicotine that mediated synchronous multicellular [Ca(2+)]i increases. In addition, nicotinic stimulation of a single cell triggered catecholamine release in coupled cells, as shown by amperometric detection of secretory events. Functional coupling between chromaffin cells in situ may represent an efficient complement to synaptic transmission to amplify catecholamine release after synaptic stimulation of a single excited chromaffin cell.

Fig. 1.

Propagation of action potential-induced [Ca²⁺]i transients between chromaffin cellsin situ. Electrical activity-driven multicellular [Ca²⁺]i increases were imaged by real-time scanning laser confocal imaging (120 images/sec with averaging 4 frames) in five chromaffin cells loaded with Oregon Green 488 BAPTA-1 as the Ca²⁺-sensitive fluorescent probe. Fluorescence emission changes were normalized to baseline fluorescenceF/F_min. The stimulated cell is indicated by an asterisk. Each image corresponds to 10 averaged confocal images before (a), during (b), and after (c) depolarization. Action potentials were triggered by injecting depolarizing current into cell 1 (V_m = −75 mV). A brief depolarization (50 msec) inducing a doublet of action potentials (A) or a sustained depolarization (500 msec) evoking a burst of 13 action potentials (B) leads to a simultaneous transient [Ca²⁺]i increase in both the stimulated and a neighboring cell (cell 2). Note that cell 5 (B) spontaneously displayed a [Ca²⁺]i change during the recording that was not linked to the action potentials evoked in cell 1.

Fig. 2.

Synchronized spontaneous [Ca²⁺]i transients between chromaffin cells.A, Spontaneous [Ca²⁺]i changes were imaged in five chromaffin cells. Plots of relative Oregon Green 488 BAPTA-1 emission changes showing synchronized [Ca²⁺]i transients in three of five cells. Note that the two other cells remained silent. B, Reversible blocking effect of a 30 sec TTX (0.5 μm) + Cd²⁺ (0.5 mm) ejection on spontaneous [Ca²⁺]i transients. Insets,Detailed kinetics of two [Ca²⁺]i transients before and after application of blockers.

Fig. 3.

Electrical coupling between chromaffin cell pairs. Membrane potential and macroscopic ionic currents were monitored in chromaffin cell pairs using the dual patch-clamp technique.A, Illustration of a cell pair in which the triggering of action potentials in the stimulated cell resulted in small membrane depolarizations in the unstepped cell. The two cells were current-clamped at −80 mV. Note that cell 2 was itself able to generate depolarization-evoked action potentials. B, Example of a cell pair in which action potentials were transmitted to the nonstimulated cell. The two cells were current-clamped at −80 mV.C, Histogram illustrating the wide distribution range of the coupling ratio calculated in 30 chromaffin cell pairs from current-clamp measurements of voltage amplitude (in response to a hyperpolarizing current injection) in both cell 1 (stepped cell) and cell 2 (target cell) (from 0 for noncoupled pairs to 1 for highly coupled pairs). The number of recorded cells is indicated inparentheses. D, Inset, Chart recordings of junctional currents (Ij) in a Cs⁺-loaded (140 mmCs⁺-gluconate) cell pair voltage clamped at −80 mV (voltage steps from −160 to +20 mV, 100 msec duration).Bottom, I–V relationship in which the junctional current amplitude (Ij) was plotted as a function of the transjunctional voltage (Vj) from −80 to +100 mV. The curves used to fit the data were derived from the linear regression y = 2.98× − 0.61 (dotted line). The correlation coefficientr² was 0.98. Three to six cell pairs were used to determine the I–V curve at each transjunctional potential.

Fig. 4.

Expression of Cx36 and Cx43 mRNA in a single chromaffin cell. The harvested cell was identified as chromaffin cell by the presence of the DβH transcript (438 bp). Gel electrophoresis of RT-PCR products of the DβH-positive cell in which Cx36 mRNA (predicted size of 360 bp) and Cx43 mRNA (425 bp) were co-detected.MW, Size marker.

Fig. 5.

Lucifer yellow diffusion between spontaneously synchronized chromaffin cells and blockade by carbenoxolone.A, Optical measurements of two neighboring cells displaying synchronized spontaneous [Ca²⁺]i transients. After fluorimetric recording, cell 1 was patched with LY (1 mm in the intrapipette solution). A few seconds later, LY diffused into cell 2. B, Pooled data summarizing the probability of observing an intercellular coupling (seen by LY diffusion) between chromaffin cells injected at random in the presence or absence of the uncoupling agent carbenoxolone (100 μm, bath-application, 10–30 min). Control and treated slices came from the same adrenal glands and results are representative of three different experiments.

Fig. 6.

Blocking effect of carbenoxolone on nicotine-induced simultaneous [Ca²⁺]i increases within a chromaffin cell cluster. Nicotine (200 mm, 1 msec) was iontophoretically applied on cell 1 through a sharp microelectrode (the onset of the nicotinic stimulation is shown by anarrow). The [Ca²⁺]i increase originating in the stimulated cell was simultaneously detected in two adjacent cells. The [Ca²⁺]i rises in the nonstimulated cells (cell 2 and cell 3) were reversibly abolished in the presence of the gap junction blocker carbenoxolone (100 μm, 15 min bath application).B, Pooled data. The number of cell clusters recorded is indicated in parentheses. *p < 0.001, as compared with control values.

Fig. 7.

Action potential-induced catecholamine release in coupled chromaffin cells. A burst of action potentials (1 sec depolarization, perforated patch-clamp) was triggered in a chromaffin cell. Subsequent catecholamine exocytosis was simultaneously monitored by the amperometry technique at a constant voltage (+800 mV) in the stepped cell (A, cell 1*) and, during a second trial, in an adjacent cell (B, cell2). As shown by the outwardly directed current deflections, action potentials were effective in stimulating catecholamine release in both the stepped cell and the neighboring cell.

Fig. 8.

Nicotinic stimulation triggers catecholamine exocytosis in coupled chromaffin cells. The iontophoretic application of nicotine (1 msec; indicated by an arrow) on cell 1* evoked a simultaneous [Ca²⁺]i rise in cell 2 but was without effect on cell 3. Catecholamine exocytosis was sequentially followed by the amperometric detection of secretory events (constant voltage of +800 mV) in cell 2 and then cell 3, respectively. As shown by outward currents, nicotine triggered catecholamine release in cell 2 only. Insets, Nicotine-induced [Ca²⁺]i rises imaged in cell 1 during amperometric recordings of cells 2 and 3.